Mhc class II haplotype specific immunodominancy of peptides derived from rsv fusion (f) and attachement (g) proteins

ABSTRACT

The present invention relates immunodominant peptides derived from human respiratory syncytial virus (H-RSV) that may be used in ex vivo diagnosis of immune responses to H-RSV The immunodominant peptides are derived from the H-RSV Fusion (F) and Attachment (G) proteins and are capable of inducing an antigen specific CD4 +  T cell response ex vivo in a MHC class 11 haplotype restricted manner. The immunodominant H-RSV-derived peptides may further be used in methods for vaccination against H-RSV, preferably in a MHC class 11 haplotype specific manner.

FIELD OF THE INVENTION

The present invention relates immunodominant peptides derived from humanrespiratory syncytial virus (H-RSV) that may be used in ex vivodiagnosis of immune responses to H-RSV, preferably in a MHC class IIhaplotype specific manner. The immunodominant peptides are preferablyderived from the H-RSV Fusion (F) and Attachment (G) proteins. Theinvention further relates to the use of these immunodominantH-RSV-derived peptides for vaccination against H-RSV, preferably in aMHC class II haplotype specific manner.

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (H-RSV), classified in the genusPneumoviruses of the family of Paramyxoviridae, is a major cause oflower respiratory disease in young infants, immunocompromisedindividuals and the elderly. No safe and effective licensed H-RSVvaccine is currently available. In an early vaccine trial with aformalin inactivated alum-precipitated H-RSV vaccine (FI-RSV) givenintramuscularly, an enhancement of disease occurred in vaccinees uponsubsequent exposure to the natural virus. This enhanced illness wascharacterised by bronchiolitis, hypoxemia and pneumonia and infiltrationinto the lungs of lymphocytes, neutrophils and some eosinophils. Thiscellular infiltration suggested that immune-mediated injury cancontribute to the pathogenicity of RSV disease.

Primary infection with RSV can cause lower respiratory tract disease inyoung infants manifesting as a pneumonia or bronchiolitis. The diseaseis associated with the inflammatory response to infection likelyinvolving the production of cytokines and chemokines by lung epithelialcells and immune cells that are recruited to the lungs. The T cellresponse is an essential component of the immune response needed forviral clearance from the lungs (Alwan et al., 1992, Clin. Exp. Immunol.8: 527-536; Graham et al., 1991, J.Clin. Invest. 88:1026-1033; FishautM. et al., 1980, J. Pediatr. 96:179-186). Antibodies against F and Gproteins are generated during RSV infection but even in the presence ofhigh levels of virus neutralising antibodies reinfections occur (Hall etal., 1991, J. Inf. Dis. 163: 693-698) and antibodies are not needed forviral clearance (Graham et al., 1991, J. Virol. 65: 4936-4942). Also,vaccination with the FI-RSV vaccine induced high titres of RSV specificantibodies in vaccinees, yet caused more severe clinical disease uponnatural infection. While in RSV infections T cell responses are requiredfor clearance of RSV from the lung it has been shown in murine studiesthat CD4 as well as CD8 T cells can be responsible for enhanced lungpathology (Cannon et al., 1988, J.Exp. Med. 168:1163-1168; Alwan et al.,1994, J. Exp. Med. 179: 81-89). Severe pneumonia with extensive influxof eosinophils into the lungs can be elicited in mice by Th2 cellsspecific for RSV-G, that are primed during vaccination with FI-RSVbefore intra-nasal challenge with life RS-virus (Waris et al., 1996, J.Virol. 70: 2852-2860; Openshaw et al., 1992, Int. Immunol. 4: 493-500).This strong eosinophilic inflammation is also observed after RSVchallenge of mice vaccinated with a vaccinia recombinant expressingsolely the G protein of RSV. One peptide corresponding to residues183-195 in the G protein of RSV appears to be recognised by Balb/cspecific Th2 cells (Varga et al., 2000, J. Immunol. 165: 6487-6495). CD8T cell responses against epitopes derived from G protein have not beenobserved in BALB/c mice nor in man. CD4 T cells in mice vaccinated witha vaccinia recombinant expressing the F protein of RSV that aresubsequently challenged with life virus are Th1 (Alwan et al., 1993, J.Immunol. 150: 5211-5218). CD8 T cell responses against F are common inmice of different MHC types (Chang et al., 2001, J. Immunol.167:4254-4260). In the Balb/c model a CD8 T cell response has been shownto regulate the outcome of CD4 T cell responses preventing enhanceddisease (Srikiatkhachorn and Braciale, 1997, J. Exp. Med. 186: 421-432).Whether the insights obtained from the murine model can be translated tothe human situation has to be evaluated.

In PBMC's from healthy adults as well as from diseased infants, CD8 Tcell responses and CD4 T cell responses can be visualised. Someinformation on viral protein/peptide recognition by human CD4 T cellsand CD8 T cells has been uncovered in the art. E.g. in Levely et al.(1991, J. Virol. 65: 3789-3796) CD4 T cell responses against somesubdomains of the F protein have been described using T cellproliferation assays. A dominant antigenic peptide was characterisedencompassing amino acid residues 338-355 of the F protein. The responseagainst this peptide was HLA-DR restricted, but the presenting MHCmolecule was not further identified. Moreover, the peptide wasrecognised by different donors that did not share one single MHC classII molecule. Goulder et al. (2000, J. Virol. 74: 7694-7697) describe anH-RSV specific cytotoxic T cell epitope that is HLA class I (HLA B7)restricted and that is derived from the H-RSV nucleoprotein. Brandenburget al. (2000, J. Virol 74: 10240-10244) describe HLA class I restrictedcytotoxic T cell epitopes derived from the H-RSV F protein. A peptidespanning amino acids 118-126 of H-RSV F protein proved to be HLA B57restricted in one patient and a peptide spanning amino acids 551-559 ofH-RSV F protein proved to be HLA C12 restricted in another patient.

There is, however, still a need for further characterisation of the MHCclass II haplotype specific immunodominancy of peptides derived from theF and G proteins of RSV. Such detailed knowledge about antigenicpeptides may be used for the development of subunit vaccines and it mayfurther enable the use of tools, like MHC class II tetramers, to monitorex vivo virus specific immune responses in infected patients or evaluatecorrelates of protection in vaccinated individuals.

DESCRIPTION OF THE INVENTION

The present invention is based on the discovery and characterisation ofimmunodominant peptides derived from the Fusion (F) and Attachment (G)proteins of H-RSV that are recognised by autologous CD4 T cells in thecontext of a panel of MHC class II molecules that are expressed mostfrequently within the Caucasian population. Antigenic peptides are foundin both G and F protein. In most donors, CD4 cells recognise more thanone peptide within the F protein. Several peptide antigens areproductively presented in the context of more than one MHC class IImolecule, while at the level of detection of the direct elispot assaysperformed, there are also peptides that are MHC class II haplotypespecific. Most peptides are presented by HLA DR molecules. In generaldonors that share MHC class II molecules recognise the same pattern ofpeptides. The present invention thus relates to diagnostic, prophylacticand therapeutic methods that are based on the MHC class II haplotypespecific antigenicity of H-RSV F or G protein-derived peptides.

In a first aspect the invention relates to method for ex vivo diagnosisof MHC class II haplotype specific immune responses to H-RSV antigens ina subject. The method comprises the steps of: (a) determining the MHCclass II haplotype of the subject; (b) providing a compositioncomprising peripheral blood mononuclear cells (PBMC's) from the subject;(c) mixing the composition comprising PBMC's with a peptide comprisingat least 9 contiguous amino acids from an amino acid sequence selectedfrom Table 1 that matches the MHC class II haplotype of the subject inaccordance with Table 1; and, (d) determining the response of the PBMC'sto the peptide.

In the method, the MHC class II haplotype of the subject is determinedusing any one of a number of methods well known in the art, (see e.g.Coligan et al., 1994, In: Coico R, ed. Current protocols in immunology.Vol. 2: John Wiley & Sons, Inc., Chapter 7: Immunologic studies inhumans). Similarly, the composition comprising PBMC's is obtained fromthe (human) subject using a variety of methods well known in the art(see e.g. Coligan et al., 1994, In: Coico R, ed. Current protocols inimmunology. Vol. 2: John Wiley & Sons, Inc.,: 711-712). Blood samplesare usually processed to remove erythrocytes and platelets (e.g., byaphaeresis, Ficoll density gradient centrifugation and/or red blood celllysis or other such methods known to one of skill in the art) and theremaining PBMC sample, which includes the T-cells of interest, as wellas B-cells, macrophages and dendritic cells, may be used directly in theassay. The composition comprising PBMC's may e.g. consist of the PBMCbulk that is obtainable by from blood obtained from the subject. Forhigher yields of PBMCs, the subject from whom the PBMCs are obtained maybe given a pre-treatment with GM-CSF for mobilising mononuclear cellsubpopulations. The PBMC composition may further be enriched forspecific subsets of mononuclear cells, preferably T cells, morepreferably CD4⁺ T cells. The PBMC composition may be enriched for Tcells, or in particular for CD4⁺ T cells, by methods known in the art,such e.g. by expansion of T cells or CD4⁺ T cells as described e.g. inColigan et al. (1994, In: Coico R, ed. Current protocols in immunology.Vol. 2: John Wiley & Sons, Inc., 1994: 711-94).

In step (c) of the diagnostic methods of the invention a selectedpeptide derived from the H-RSV F and/or G proteins is mixed with thecomposition comprising the PBMC's. The H-RSV F or G protein-derivedpeptides of the invention may be obtained as described below. Thepeptide to be mixed with the composition comprising the PBMC's of agiven subject are selected to match the MHC class II haplotype of thesubject. Table 1 summarises for each of the major MHC class IIhaplotypes that are prevalent in the caucasian population, the H-RSV Fand G protein derived peptides that are recognised by the particular MHCclass II haplotype, as measured by IFN-γ production in a direct elispotassay. Thus, in the method of the invention, subsequent to havingestablished the MHC class II haplotype of a subject, the compositioncomprising the PBMC's form the a subject is brought into contact withone or more (poly)peptides that comprise an amino acid sequence of atleast 9, 10, 11 or 12 contiguous amino acids from an amino acid sequencelisted in Table 1 as being recognised by the MHC class II haplotype ofthe subject. Peptides and polypeptides of the invention are furtherdescribed herein below. Preferably the peptides are brought into contactwith the composition comprising the PBMC's at a concentration thatranges from 1 nM to 100 μM, more preferably 10 nM to 50 μM, mostpreferably 100 nM to 10 μM. The amount of PBMC's to be brought intocontact with the peptide depends on the assay format used for detectingthe PBMC response. Various such assays are described below and theamount of PBMC's to be used in these assays are known to the skilledperson.

Finally, in step (d) of the method, the response of the PBMC's to theH-RSV antigenic peptide(s) is determined. The antigen specific responseof the PBMC's may be determined using a number of methods available inthe art (see e.g. Coligan et al., 1994, In: Coico R, ed. Currentprotocols in immunology. Vol. 2: John Wiley & Sons, Inc., Chapter 7:Immunologic studies in humans). Preferably, the antigen specificresponse of the PBMC's is determined by determining the proliferation ofthe PBMC's in response to the presence of the H-RSV antigenic peptides.More preferably, the proliferation of T cells in response to thepresence of the H-RSV peptide(s) is determined. The proliferation of Tcells in response to the presence of the H-RSV peptide(s) is preferablydetermined directly in the composition comprising the PBMC's, i.e.without pre-expansion of the T cells. Alternatively, the T cells or morespecifically, the CD4⁺ T cells may be enriched or pre-expanded toincrease the sensitivity of the diagnostic method of the invention. In afurther preferred method, the proliferation of CD4⁺ T cells (in thecomposition comprising PBMC's) in response to the presence of the H-RSVpeptide(s) is determined. The proliferation or response of the CD4⁺ Tcells may be determined using a variety of methods available in the art,(see e.g. Coligan et al., 1994, In: Coico R, ed. Current protocols inimmunology. Vol. 2: John Wiley & Sons, Inc., Chapter 7: Immunologicstudies in humans). In a preferred method the proliferation or responseof the PBMC's, in particular the CD4⁺ T cells is determined by measuringthe concentration of a “soluble protein factor” that is secreted by aPBMC or T-cell in response to antigenic stimulation. A variety ofsecreted soluble protein factors can be detected by the assays disclosedherein. The soluble factors may be cytokines, lymphokines or chemokines.Typically this secreted factor is a lymphokine, such as enumeratedbelow. As a result of the increased sensitivity of the assay, factorssecreted by rare T-cells which occur in low frequency can be detected.Factors which are detected by this method include, but are not limitedto lymphokines, cytokines and chemokines such as for example, IFN-γ,TNF-α, IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, TGF-β, RANTES, and GM-CSF.

A variety of assay formats can be used to detect the increased levels ofsecreted factors produced by the assay described herein. Suitable assaysinclude both solid phase (heterogeneous) and non-solid phase(homogeneous) protocols. The assays can be run using competitive ornon-competitive formats, and using a wide variety of labels, such asradioisotopes, enzymes, fluorescers, chemiluminescers, spin labels, andthe like. Such methods include, but are not limited to enzyme-linkedimmunosorbent assays (ELISA), both direct and reverse formats, and othersolid phase assays. It will be recognised that negative controls, i.e.,samples run without added antigen, and positive controls, i.e., samplesrun with antigens, such as tetanus toxoid, known to elicit lymphokinesecretion from T-cells will be run as necessary under otherwiseduplicative conditions to validate the assay results.

Some assays rely on heterogeneous protocols where a ligand complementaryto the secreted factor (such as antibody against the secreted factor) isbound to a solid phase which is used to capture the secreted factor. Theligand may be conveniently immobilised on a variety of solid phases,such as dipsticks, particulates, microspheres, magnetic particles, testtubes, microtiter wells, and plastics, nitrocellulose or nylon membranesand the like, including polyvinyl difluoride (PVDF) (e.g., 96 well platewith a PVDF membrane base (Millipore MAIPS45-10))and ELISA gradeplastic. The captured factor can then be detected using thenon-competitive “sandwich” technique where a directly or indirectlylabelled second ligand for the factor is exposed to the washed solidphase. Such assay techniques are well known and well described in boththe patent and scientific literature. See, e.g., U.S. Pat. Nos.3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876. Enzyme-linked immunosorbent assay (ELISA)methods are described in detail in U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,879,262; and 4,034,074. ELISA assays detect very low titresof secreted factors. Also see, “Enzyme immunohistochemistry” in Practiceand Theory of Enzyme Immunoassays, P. Tijssen (Elsevier 1985).

A commonly used assay format is the antibody capture assay. The generalprotocol is simple: a ligand, e.g., an unlabeled antibody for thesecreted factor, is immobilised on a solid phase, and the secretedfactor is allowed to bind to the immobilised antibody. The boundsecreted factor is then detected by using a labelled secondary reagentthat will specifically bind to the captured factor (“direct sandwichassay”). Alternatively, the secondary reagent will not be labelled, butwill be detected by subsequent binding to labelled tertiary bindingreagent complementary to the second binding reagent (“indirect sandwichassay”). The strength of signal from the bound label allows thedetermination of the amount of secreted factor present in the sample andthis in turn allows the quantification of the number of activatedT-cells present in the sample. As will be recognised by the skilledperson, the sandwich assay can be used to detect any secreted factorwhich has two epitopes, each of which can be recognised by the specificbinding pair members. Choosing appropriate capture and detectionantibody pairs permits the application of this assay to the detection ofT cells secreting a variety of soluble factors. A list of antibody pairswhich can be used in this assay is inter alia presented in U.S. Pat. No.6,218,132.

Alternatively, the proliferation or response of the T cells may bedetermined by intracellular cytokine staining as e.g. described byMurali-Krishna et al. (1998, Immunity 8: 177-187).

A preferred method for determining the proliferation or response of theCD4⁺ T cells comprises measuring the IFN-γ production by the CD4⁺ Tcells in response to the presence of the H-RSV peptide(s). Again,methods for measuring antigen induced IFN-γ production by the CD4⁺ Tcells are well known in the art (see e.g. Coligan et al., 1994, In:Coico R, ed. Current protocols in immunology. Vol. 2: John Wiley & Sons,Inc., Chapter 6: Cytokines)., and include e.g. a method wherein IFN-γproduction is measured in a elispot assay, as described in the Examplesherein. Preferably, the IFN-γ production is measured in a direct elispotassay, whereby “direct” is understood to mean directly on thecomposition of PBMC's obtained from the subject, without pre-expansionof the T cells or CD4⁺ T cells. Likewise, the other above-mentionedlymphokines and cytokines may be measured in a direct elispot assay.

The diagnostic methods of the invention may be used for a variety ofreasons on different subjects. The methods may e.g. be used on a subjectundergoing a lower respiratory tract (LRT) disease as manifested bypneumonia and/or bronchitis. In such subjects, the method may be used todetermine the involvement of H-RSV in the LRT disease, e.g. todistinguish between infections with H-RSV, influenza virus and/or humanpneumovirus. Having established the diagnosis of an H-RSV infection in agiven subject, the diagnostic methods of the invention may further beused to monitor the immune response to H-RSV antigens in the subjectundergoing an H-RSV infection. The methods may further be used todetermine the status of a subject's immune response to H-RSV in asubject having undergone an infection with H-RSV, i.e. in a subject thatno longer shows any clinical symptoms of H-RSV infection. Finally, themethods of the invention may be used to determine the status of asubject's immune response to H-RSV in a subject having been vaccinatedagainst H-RSV. In particular, the methods may be used to evaluatecorrelates of protection in individuals having been vaccinated againstH-RSV.

In a further aspect, the invention relates to a method for MHC class IIhaplotype specific vaccination of a subject against H-RSV, the methodcomprising the steps of: (a) determining the MHC class II haplotype ofthe subject; and, (b) administering to the subject a pharmaceuticalcomposition comprising a peptide comprising at least 9, 10, 11, or 12contiguous amino acids from an amino acid sequence selected from Table1, whereby the amino acid sequence matches the MHC class II haplotype ofthe subject in accordance with Table 1. In this method, the MHC class IIhaplotype of the subject is determined by known methods as describedabove. Method for preparing the peptides to be used in this method, aswell as pharmaceutical composition comprising these peptides and methodsfor their preparation are described herein below. In a preferred methodof vaccination, a pharmaceutical composition suitable for parenteraladministration is administered parenterally, a pharmaceuticalcomposition suitable for transdermal administration is administeredtransdermally, or a pharmaceutical composition suitable for inhalationis inhaled.

In yet another aspect, the invention relates to the use of a peptidecomprising at least 9, 10, 11, or 12 contiguous amino acids from anamino acid sequence selected from Table 1, for the manufacture of avaccine for MHC class II haplotype specific prophylaxis or therapy ofH-RSV infection in a subject, whereby the amino acid sequence matchesthe MHC class II haplotype of the subject in accordance with Table 1.Preferably, the vaccine is a pharmaceutical composition suitable forparenteral or transdermal administration.

Peptides for Use in the Methods of the Invention

The peptides of the invention contain an epitope that specificallyrecognised by MHC class II molecules in accordance with Table 1. Thepeptides of the invention thus bind the groove or cleft of the MHC classII molecule in question. The peptides of the invention typicallycomprise at least about 9, 10, 11, 12, 15, or 18 residues. In certainembodiments the peptides will not exceed about 150, 100 or 50 residuesand typically will not exceed about 20 residues. Thus, a wide range ofpeptide sizes may be used in the present invention.

Particularly because the peptides to be used in the methods of theinvention are relatively short, the peptides can be readily synthesisedusing known methods. For example, the peptides can be synthesised by thewell-known Merrifield solid-phase synthesis method in which amino acidsare sequentially added to a growing chain. See Merrifield (1963), J. Am.Chem. Soc. 85:2149-2156; and Atherton et al., “Solid Phase PeptideSynthesis,” IRL Press, London, (1989). Automatic peptide synthesisersare commercially available from numerous suppliers, such as AppliedBiosystems, Foster City, Calif. Additional synthetic approaches forpreparing the peptides of the invention are described in the Examplesherein.

Alternatively, the F and G protein-derived peptides may be prepared aspart of larger polypeptides comprising one or more of the peptides ofthe invention. Such larger polypeptides be prepared using well-knownrecombinant techniques in which a nucleotide sequence encoding thepolypeptide of interest is expressed in cultured cells such as describedin Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York (1987) and in Sambrook andRussell (2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NewYork, both of which are incorporated herein by reference in theirentirety. Also see, Kunkel, 1985, Proc. Natl. Acad. Sci. 82:488(describing site directed mutagenesis) and Roberts et al., 1987, Nature328:731-734 or Wells, J. A., et al. (1985) Gene 34:315 (describingcassette mutagenesis).

Typically, nucleic acids encoding the desired polypeptides are used inexpression vectors. The phrase “expression vector” generally refers tonucleotide sequences that are capable of affecting expression of a genein hosts compatible with such sequences. These expression vectorstypically include at least suitable promoter sequences and optionally,transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used as described herein.DNA encoding the polypeptides of the present invention will typically beincorporated into DNA constructs capable of introduction into andexpression in an in vitro cell culture. Specifically, DNA constructswill be suitable for replication in a prokaryotic host, such asbacteria, e.g., E. coli, or may be introduced into a cultured mammalian,plant, insect, yeast, fungi or other eukaryotic cell lines.

DNA constructs prepared for introduction into a particular host willtypically include a replication system recognised by the host, theintended DNA segment encoding the desired polypeptide, andtranscriptional and translational initiation and termination regulatorysequences operably linked to the polypeptide encoding segment. A DNAsegment is “operably linked” when it is placed into a functionalrelationship with another DNA segment. For example, a promoter orenhancer is operably linked to a coding sequence if it stimulates thetranscription of the sequence. DNA for a signal sequence is operablylinked to DNA encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide. Generally, DNAsequences that are operably linked are contiguous, and, in the case of asignal sequence, both contiguous and in reading phase. However,enhancers need not be contiguous with the coding sequences whosetranscription they control. Linking is accomplished by ligation atconvenient restriction sites or at adapters or linkers inserted in lieuthereof.

The selection of an appropriate promoter sequence generally depends uponthe host cell selected for the expression of the DNA segment. Examplesof suitable promoter sequences include prokaryotic, and eukaryoticpromoters well-known in the art. See, e.g., Sambrook and Russell (2001,supra). The transcriptional regulatory sequences will typically includea heterologous enhancer or promoter which is recognised by the host. Theselection of an appropriate promoter will depend upon the host, butpromoters such as the trp, lac and phage promoters, tRNA promoters andglycolytic enzyme promoters are known and available. See, e.g., Sambrookand Russell (2001, supra).

Conveniently available expression vectors which include the replicationsystem and transcriptional and translational regulatory sequencestogether with the insertion site for the polypeptide encoding segmentmay be employed. Examples of workable combinations of cell lines andexpression vectors are described in Sambrook and Russell (2001, supra).For example, suitable expression vectors may be expressed in, e.g.,insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells andbacterial cells, e.g., E. coli.

It will be understood that the H-RSV F or G protein derived(poly)peptides of the invention may be modified to provide a variety ofdesired attributes, e.g., improved pharmacological characteristics,while increasing or at least retaining substantially all of thebiological activity of the unmodified peptide. For instance, thepeptides can be modified by extending, decreasing the amino acidsequence of the peptide. Substitutions with different amino acids oramino acid mimetics can also be made.

The individual residues of the immunogenic H-RSV F or G protein derived(poly)peptides of the invention can be incorporated in the peptide by apeptide bond or peptide bond mimetic. A peptide bond mimetic of theinvention includes peptide backbone modifications well known to thoseskilled in the art. Such modifications include modifications of theamide nitrogen, the a-carbon, amide carbonyl, complete replacement ofthe amide bond, extensions, deletions or backbone cross-links. See,generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Vol. VII (Weinstein ed., 1983). Several peptide backbonemodifications are known, these include, ψ [CH₂S], ψ [CH₂NH], ψ [CSNH₂],ψ [NHCO], ψ [COCH₂ ] and ψ [(E) or (Z) CH═CH]. The nomenclature usedabove, follows that suggested by Spatola, above. In this context, ψindicates the absence of an amide bond. The structure that replaces theamide group is specified within the brackets.

Amino acid mimetics may also be incorporated in the peptides. An “aminoacid mimetic” as used here is a moiety other than a naturally occurringamino acid that conformationally and functionally serves as a substitutefor an amino acid in a peptide of the present invention. Such a moietyserves as a substitute for an amino acid residue if it does notinterfere with the ability of the peptide to elicit an immune responseagainst the appropriate H-RSV F or G protein derived epitope. Amino acidmimetics may include non-protein amino acids, such as β-, γ-, δ-aminoacids, β-, γ-, δ-imino acids (such as piperidine-4-carboxylic acid) aswell as many derivatives of L-α-amino acids. A number of suitable aminoacid mimetics are known to the skilled artisan, they includecyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine,adamantylacetic acid and the like. Peptide mimetics suitable forpeptides of the present invention are discussed by Morgan and Gainor,(1989) Ann. Repts. Med. Chem. 24:243-252.

As noted above, the peptides employed in the subject invention need notbe identical, but may be substantially identical, to the correspondingamino acid sequence of the H-RSV F or G protein derived epitope aslisted in Table 1. Therefore, the peptides may be subject to variouschanges, such as insertions, deletions, and substitutions, eitherconservative or non-conservative, where such changes might provide forcertain advantages in their use. The peptides of the invention can bemodified in a number of ways so long as they comprise a sequencesubstantially identical (as defined below) to an amino acid sequence ofan H-RSV F or G protein derived epitope as listed in Table 1.

Alignment and comparison of relatively short amino acid sequences (lessthan about 30 residues) is typically straightforward. Optimal alignmentof sequences for aligning a comparison window may be conducted by thelocal homology algorithm of Smith and Waterman (1981) Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, bycomputerised implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection, and the best alignment (i.e., resulting in the highestpercentage of sequence similarity over the comparison window) generatedby the various methods is selected.

The term “sequence identity” means that two polypeptide sequences areidentical (i.e., on an amino acid-by-amino acid basis) over a window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical residuesoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

As applied to the peptides of the invention, the term “substantialidentity” means that two peptide sequences, when optimally aligned, suchas by the programs GAP or BESTFIT using default gap weights, share atleast 80 percent sequence identity, preferably at least 90 percentsequence identity, more preferably at least 95 percent sequence identityor more (e.g., 99 percent sequence identity). Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulphur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Pharmaceutical (Vaccine) Compositions and their Administration

The peptides of the present invention and pharmaceutical compositionsthereof are useful for administration to mammals, particularly humans,to treat and/or prevent H-RSV infection as well as LRT and otherdiseases caused by H-RSV infection. Suitable formulations are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985), which is incorporated herein byreference.

The immunogenic peptides of the invention are administeredprophylactically or to an individual already suffering from the disease.The compositions are administered to a patient in an amount sufficientto elicit an effective immune response to the MHC molecule from whichthe peptides are derived. An amount adequate to accomplish this isdefined as “therapeutically effective dose” or “immunogenicallyeffective dose.” Amounts effective for this use will depend on, e.g.,the peptide composition, the manner of administration, the stage andseverity of the disease being treated, the weight and general state ofhealth of the patient, and the judgement of the prescribing physician,but generally range for the initial immunisation (that is fortherapeutic or prophylactic administration) from about 0.1 mg to about1.0 mg per 70 kilogram patient, more commonly from about 0.5 mg to about0.75 mg per 70 kg of body weight. Boosting dosages are typically fromabout 0.1 mg to about 0.5 mg of peptide using a boosting regimen overweeks to months depending upon the patient's response and condition. Asuitable protocol would include injection at time 0, 2, 6, 10 and 14weeks, followed by booster injections at 24 and 28 weeks.

The pharmaceutical compositions are intended for parenteral, oral ortransdermal administration. Preferably, the pharmaceutical compositionsare administered parenterally, e.g., subcutaneously, intradermally, orintramuscularly. Thus, the invention provides compositions forparenteral administration which comprise a solution of the immunogenicpeptides dissolved or suspended in an acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilised by conventional, well knownsterilisation techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilised, thelyophilised preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,and triethanolamine oleate.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%. As noted above, thecompositions are intended to induce an immune response to the peptides.Thus, compositions and methods of administration suitable for maximisingthe immune response are preferred. For instance, peptides may beintroduced into a host, including humans, linked to a carrier or as ahomopolymer or heteropolymer of active peptide units. Alternatively, thea “cocktail” of peptides can be used. A mixture of more than one peptidehas the advantage of increased immunological reaction and, wheredifferent peptides are used to make up the polymer, the additionalability to induce antibodies to a number of epitopes. For instance,peptides comprising sequences from hypervariable regions of α and βchains may be used in combination. Useful carriers are well known in theart, and include, e.g., thyroglobulin, albumins such as human serumalbumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamicacid), influenza, hepatitis B virus core protein, hepatitis B virusrecombinant vaccine and the like.

The compositions preferably also include an adjuvant. A number ofadjuvants are well known to one skilled in the art. Suitable adjuvantsinclude incomplete Freund's adjuvant, alum, aluminum phosphate, aluminumhydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicpeptide. A particularly useful adjuvant and immunisation schedule aredescribed in Kwak et al. New Eng. J. Med. 327-1209-1215 (1992), which isincorporated herein by reference. The immunological adjuvant describedthere comprises 5% (wt/vol) squalene, 2.5% Pluronic L121 polymer and0.2% polysorbate in phosphate buffered saline.

The concentration of immunogenic peptides of the invention in thepharmaceutical formulations can vary widely, i.e. from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

The peptides of the invention can also be expressed by attenuated viralhosts, such as vaccinia or fowlpox. This approach involves the use ofvaccinia virus as a vector to express nucleotide sequences that encodethe peptides of the invention. Upon introduction into a host, therecombinant vaccinia virus expresses the immunogenic peptide, andthereby elicits an immune response. Vaccinia vectors and methods usefulin immunisation protocols are described in, e.g., U.S. Pat. No.4,722,848, incorporated herein by reference. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.(Nature 351:456-460 (1991)) which is incorporated herein by reference. Awide variety of other vectors useful for therapeutic administration orimmunisation of the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art fromthe description herein.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990). Both of these references are incorporated hereinby reference in their entirety. E.g. transdermal delivery systemsinclude patches, gels, tapes and creams, and can contain excipients suchas solubilisers, permeation enhancers (e.g. fatty acids, fatty acidesters, fatty alcohols and amino acids), hydrophilic polymers (e.g.polycarbophil and polyvinyl pyrillidine and adhesives and tackifiers(e.g. polyisobutylenes, silicone-based adhesives, acrylates andpolybutene). Transmucosal delivery systems include patches, tablets,suppositories, pessaries, gels, and creams, and can contain excipientssuch as solubilisers and enhancers (e.g. propylene glycol, bile saltsand amino acids), and other vehicles (e.g. polyethylene glycol, fattyacid esters and derivatives, and hydrophilic polymers such ashydroxypropylmethyl cellulose and hyaluronic acid). Enjectable deliverysystems include solutions, suspensions, gels, microspheres and polymericinjectables, and can comprise excipients such as solubility-alteringagents (e.g. ethanol, propylene glycol and sucrose) and polymers (e.g.polycaprylactones, and PLGA's). Implantable systems include rods anddiscs, and can contain excipients such as PLGA and polycapryl lactone.Other delivery systems that can be used for administering thepharmaceutical composition of the invention include intranasal deliverysystems such as sprays and powders, sublingual delivery systems andsystems for delivery by inhalation. For administration by inhalation,the pharmaceutical compositions of the present invention areconveniently delivered in the form of an aerosol spray presentation frompressurised packs or a nebuliser, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurised aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the peptides of the invention and a suitablepowder base such as lactose or starch. The pharmaceutical compositionsof the invention may be further formulated for administration byinhalation as e.g. described in U.S. Pat. No. 6,358,530.

In another aspect the invention relates to a method for producing apharmaceutical composition comprising the (poly)peptides of theinvention. The method comprises at least the steps of mixing the(poly)peptides of the invention obtained in the methods described abovewith a pharmaceutically acceptable carrier and further constituents likeadjuvant as described above.

EXAMPLES

1. Material and Methods

1.1. Peripheral Blood Mononuclear Cells (PBMC's)

Buffycoats were obtained from healthy adult blood donors with informedconsent. PBMC's were isolated by density centrifugation using lymphoprep(Nycomed Pharma). PBMC's were either thawed from cryopreserved samples(−135° C.), in RPMI 1640 (Gibco), 10% Dimethyl Sulfoxide, 30% fetalbovine serum (FBS, Gibco), or used fresh.

1.2 Virus and Peptides

Human RSV strain A2 (available under number VR 1302 from the ATCC,Manassas, Va., USA) was propagated in HEp-2 cells and titrated by plaqueassay. The virus was routinely used in experiments at a MOI of 1(MOI=multiplicity of infection). A series of 94 peptides, 18 amino acidresidues long was synthesised by standard solid phase Fmoc chemistry.All peptides were analysed by mass spectrometry and gave the anticipated(M+H)⁺ except for peptides 11,12 and 85, that were excluded from thestudy. Peptides 69 and 70 were made at the end of the series and werenumbered 95 and 96 respectively. The peptides synthesised were based onthe sequence of the fusion protein F of RSV strain A2. They overlappedby 12 amino acid residues. For the G protein we used a series overoverlapping peptides representing amino acid residues 161-199 of the Gprotein of RSV strain A2. The sequences of these G protein-derivedpeptides were: DFHFEVFNFVPCSI; FEVFNFVPCSICSNN; FNFVPCSICSNNPTCW;SICSNNPTCWAICKRI; SNNPTCWAICKRIPNKKP; WAICKRIPNKKPGKK; andICKRIPNKKPGKKTT.

1.3 Elispot Assay

96 well filtration plates (MAIPS45 10, Millipore) were coated overnightwith anti-IFN-γ coating antibody (Ab) 1-D1K (100 μl, 15 μg/ml, Mabtech)in 0.1M carbonate-bicarbonate buffer pH 9.6, at 4° C. Before adding thecells the plate were washed thoroughly with PBS, blocked for one hour at37° C. with RPMI 1640 containing 10% FBS. Cells and either virus orpeptide were added to the well in a final volume of 200 μl of RPMI 1640,10% FBS, penicillin and streptomycin. Cells were incubated at 37° C.overnight in a humidified incubator. Then cells were removed bythoroughly washing in PBS and 100 μl of detecting mAb 7-B6-1-biotin(Mabtech), diluted to 1 μg/ml in PBS-0.5% FBS was added to the wells.After incubating 2 hrs at room temperature and washes in PBS, 100 μl(1/1000 diluted in PBS, 0.5% FBS) ExtraAvidine Alkaline PhosphataseConjugate (Sigma) was added and incubated 1-2 hours at room temperature.Then the plates were washed in PBS and BCIP/NBT substrate was added 100μl/well, (BCIP/NBT tablets Sigma, 1 tablet diluted in 10 ml H₂O). Spotswere counted by two investigators. Most data are represented as thenumber of spots per 10⁻⁶ PBMC minus background of unstimulated wells.Only in Table 1 the uncorrected number of spots is shown. In the mAbblocking experiments the antibodies were added to the cells prior to thestimulating peptides. The cells and mAb were preincubated for 30 minutesat 37° C., after which the peptides were added to the cultures. The mAbused are culture supernatants of hybridoma's B8.11.2 producing antiHLA-DR and SPVL3 anti HLA-DQ.

2. Immunodominancy of F Protein Derived Peptides

A set of overlapping peptides 18 amino acid residues long andoverlapping by 12 amino acid residues, spanning the entire length of theF protein of RSV strain A2 was synthesised. In a preliminary search forantigenic epitopes pools of 6 consecutive peptides were made. Thesepeptide pools were analysed in an elispot assay for their ability toinduce IFN-γ production in PBMC from healthy donors. In Table 1 the dataare summarised for a group of donors in which the most frequentlyoccurring MHC class II molecules within the caucasian population arerepresented. We were able to detect IFN-γ production in response tostimulation with intact RSV at a MOI of 1. The level of the responsevaried from donor to donor but was generally in the order of 100 to 500spots per 10⁻⁶ PBMC. There was no correlation between better responsesand the time of the year the blood was sampled. Most donors responded tomore than one peptide pool. Pool four was not recognised by any donor.The peptides represented in this pool cover a stretch of 46 amino acidresidues from 121 to 168, partly overlapping the 27 amino acid residuepeptide (110-136) that is removed from the fusion protein after furincleavage in the Golgi-compartment (Gonzalez-Ryes et al., 2001, Proc.Natl. Acad. Sci. USA 98: 9859-9864).

In a second step the individual peptides from the positive pools thatwere able to induce IFN-γ production were identified for five individualdonors (data not shown). For most pools a clearcut response identifiedone or two positive peptides. Sometimes two adjacent peptides were foundpositive that possibly contained an overlapping epitope. The peptidelength of 18 amino acid residues is sufficiently long to encompass MHCclass II peptides and less suitable for MHC class I interaction. Forpresentation by MHC class I molecules the 18-mer peptides requirecleavage to 9-11 amino acid residues in order to be able to bind intothe class I binding groove. Yet in our initial screens we used fairlyhigh concentrations of peptides that had not been purified by HPLC.Therefore, it could be possible that the responses we observed wereeither CD4 or CD8 T cell responses. To determine the T cell subset—CD4or CD8—responding to the peptides, we performed elispot assays usingPBMC depleted for either CD8 T cells or CD4 T cells. For all thepeptides that we have tested with subset depleted PBMC we found that theresponses where completely abrogated after CD4 depletion (data notshown). The CD4 involvement in the peptide specific responses wasfurther confirmed by peptide stimulations of PBMC in the presence of HLADR blocking antibody (Ab) B8.11.2 or HLA DQ blocking Ab SPVL3 (data notshown). For most peptides the number of IFN-γ producing cells dropped tozero when the peptide concentration was titrated in the presence of MHCclass II blocking Ab B8.11.2 (data not shown). However, in two donorswith HLA type DRB1*1302, DR15, DQ6 and DRB1*1301, DQ6 the responseagainst peptides 50 and 51 is DQ restricted (data not shown). Thesepeptides are also recognised in a DQ restricted fashion by a donorexpressing HLA alleles DR1, DQ5 (data not shown). A third donor (HLADR15,9 DQ3,6) recognised two adjacent peptides (peptide numbers 45,46)also in the context of HLA-DQ (data not shown). Another observation madefrom these experiments is that there appeared to be large differences inthe minimal peptide concentration (40 nM-5 μM necessary for optimal Tcell activation for the different individual peptides. Yet the fact thatwe observed the responses means that during a natural infection thetotal array of peptides that we have detected in the present series oftests can be functionally presented to the immune system, since we couldpick up the response without pre-expansion of the T cells in a directelispot assay. To determine the MHC restriction element for a final setof peptides recognised by donors carrying HLA-DR7,11,15 alleles, antigenpresenting cells (APC) were depleted from high responder heterozygousdonors. Elispot assays were performed using these APC-depleted PBMC asresponders and fixed PBMC from other homozygous donors as APC. It wasfound that HLA-DR7 presents peptides 6, 10, 39 and 91 and HLA-DR11presents peptide 39 to CD4 cells.

From the data obtained in the second step described above, we selected aset of 30 peptides that were recognised by at least one donor. In Table1 we summarise the T cell responses against this set of F peptides for aseries of donors in which the most frequently occurring HLA-DR typeswithin the caucasian population are represented (Knipper et al., 2000,Human Immunol. 61: 605-614). For some peptides the possible P1 anchorresidue is marked, based on known antigen binding motifs or computeralgorithms (Sturniolo et al., 1999, Nature Biotech. 17: 555-561;Rammensee et al., 1997, Molecular Biology Intelligence Unit, MHC ligandsand peptide motifs, Springer Verlag, Heidelberg Germany, Int. ISBN3-540-63125-9).

3. Immunodominancy of G Protein Derived Peptides

We used the same procedure that was employed for the characterisation ofimmunodominant peptides within the Fusion protein of RSV to characteriseimmunodominant peptides derived from the G protein of RSV. Because ofthe high variability of the G protein in different virus isolates wefocussed our analysis on a conserved region (amino acid residues161-199) within G that possibly plays a role in viral attachment to thecell. Within this region we have characterised two peptides (seetable 1) that can be recognised by CD4 T cells present in PBMC samplesof healthy blood donors. TABLE 1 SEQ Peptide RSV ID MHC haplotype numberprotein Aa position No. Amino Acid sequence HLA-DR1 2 F  7-24 3KANAITILTAVTFCFAS 3 13-30 4 TILTAVTFCFASGQNITE 40 235-252 12REFSVNAGVTTPVSTYML 77 457-474 20 YYVNKQEGKSLYVKGEPI HLA-DQ5 50 295-31215 EVLAYVVQLPLYGVIDTP 51 301-318 16 VQLPLYGVIDTPCWKLHT HLA-DR4 2 F  7-243 KANAITILTAVTFCFAS (DRB1*0401) 5 25-42 5 GQNITEEFYQSTCSAVSK 6 31-48 6EFYQSTCSAVSKGYLSAl 14 79-96 8 IKQELDKYKNAVTELQLL 15 85-102 9KYKNAVTELQLLMQSTPP 66 391-408 18 YDCK I MTSKTDVSSSVIT 72 427-444 19 KNRGI IKTFSNGCDYVSN HLA-DR7 5 F 25-42 5 GQNITEEFYQSTCSAVSK 6 31-48 6EFYQSTCSAVSKGYLSAL 10 55-72 7 SVITIELSNIKENKCNGT 15  85-102 9KYKNAVTELQLLMQSTPP 30 175-192 10 NKAV V SLSNGVSVLTSKV 39 229-246 11RLLEITREFSVNAGVTTP 95 409-426 22 SLGAIVSCYGKTKCTASN 91 541-558 21LIAVGLLLYCKARSTPVT HLA-DR9 45 F 265-282 13 PITNDQKKLMSNVQIVR 46 271-28814 KLMSNNVQIVRQQSYSI HLA-DR11 39 F 229-246 11 RLLEITREFSVNAGVTTP 57337-354 17 TDRGWYCDNAGSVSFFPQ HLA-DR13 57 F 337-354 17TDRGWYCDNAGSVSFFPQ (DRB1*1301) HLA-DQ6 50 295-312 15 EVLAYVVQLPLYGVIDTP51 301-318 16 VQLPLYGVIDTPCWKLHT HLA-DR15, 51 14 F 79-96 8IKQELDKYKNAVTELQLL 95 409-426 22 SLGAIVSCYGKTKCTASN HLA-DPw4 G 162-17523 DFHFEVFNFVPCSI (DPB1*0401) and (DPB1*0402) HLA-DR G 177-194 24SNNPTCWAICKRIPNKKP

1. A method for ex vivo diagnosis of an MHC class II haplotype-specificimmune response to an H-RSV antigen in a subject, comprising the stepsof: (a) determining the MHC class II haplotype of the subject; (b)incubating peripheral blood mononuclear cells (PBMC's) from the subjectwith a peptide comprising any one or more of amino acid sequencesdesignated SEQ ID NO:3-SEQ ID NO:24, wherein the amino acid sequence isknown to be presented by the MHC class II molecules of the subject asindicated in Table 1, said incubating being under conditions whereinproliferation of, and/or cytokine production by, PBMC's is induced; and,(c) assaying the proliferation and/or cytokine production.
 2. A methodaccording to claim 1, wherein in step (c) the proliferation of, orcytokine production by, T cells is assayed.
 3. A method according toclaim 2, wherein the T cell proliferation or cytokine production isassayed without pre-expansion of the T cells.
 4. A method according toclaim 2, wherein proliferation of, or cytokine production by, CD4⁺ Tcells is assayed.
 5. A method according to claim 4, wherein the cytokineassayed is IFN-γ.
 6. A method according to claim 5, wherein IFN-γproduction is measured in an Elispot assay.
 7. A method according toclaim 1, wherein in step (b) the peptide is incubated at a concentrationof a least 5 nM.
 8. A method according to claim 1, wherein (a) thesubject is one who: (i) is or was infected with H-RSV or (ii) has beenvaccinated against H-RSV; and (b) an immune response to an H-RSV antigenmeasured.
 9. A method according claim 8, wherein the a subject has beenvaccinated against H-RSV.
 10. A method to evaluate the correlation ofprotection against H-RSV infection with vaccination in a subject,comprising (a) measuring a response of proliferation or cytokineproduction to an H-RSV peptide of the subject's PBMC's according toclaim 1, (b) examining the vaccination status of the subject, and (c)correlating said response in (a) with said vaccination status in (b).11. A method for immunizing a subject against an H-RSV antigen in an MHCclass II haplotype-specific manner, comprising: (a) determining the MHCclass II haplotype of the subject; and, (b) administering to the subjectan immunogenic pharmaceutical composition comprising a peptidecomprising any one or more of amino acid sequences designated SEQ IDNO:3-SEQ ID NO:24, wherein the amino acid sequence is known to bepresented by the MHC class II molecules of the subject as indicated inTable 1, thereby immunizing the subject in said MHC class IIhaplotype-specific manner.
 12. A method according to claim 11, whereinthe composition is: (a) formulated for parenteral administration andadministered parenterally, or (b) formulated for transdermaladministration and administered transdermally.
 13. A method forpreventing or treating H-RSV infection in a subject, comprising, beforeor during said infection, immunizing the subject in accordance withclaim 11, thereby preventing or treating said infection.
 14. The methodaccording to claim 13 wherein the immunogenic composition is formulatedfor parenteral or transdermal administration.
 15. A method according toclaim 3, wherein proliferation of, or cytokine production by, CD4⁺ Tcells is assayed.
 16. A method according to claim 15, wherein thecytokine assayed is IFN-γ.
 17. A method according to claim 3, wherein(a) the subject is one who: (i) is or was infected with H-RSV or (ii)has been vaccinated against H-RSV, and (b) an immune response to anH-RSV antigen is measured.
 18. A method according to claim 17, whereinsubject has been vaccinated against H-RSV.
 19. A method according toclaim 5, wherein (a) the subject is one who: (i) is or was infected withH-RSV or (ii) has been vaccinated against H-RSV, and (b) an immuneresponse to an H-RSV antigen is measured.
 20. A method according toclaim 19, wherein subject has been vaccinated against H-RSV.